[Activity of butenafine against ocular pathogenic filamentous fungi in vitro].

OBJECTIVE: To investigate antifungal activity of butenafine in comparison with that of natamycin, amphotericin B and fluconazole against ocular pathogenic filamentous fungi in vitro. METHODS: It was an experimental study. Susceptibility tests were performed against 260 isolates of ocular pathogenic filamentous fungi by broth dilution antifungal susceptibility test of filamentous fungi approved by the Clinical and Laboratory Standards Institute (CLSI) M38-A document. The isolates included Fusarium spp. (136), Aspergillus spp. (98), Alternaria alternata (9), Curvularia lunata (3), and unusual ocular pathogens (14). Final concentration ranged from 0.008 to 16.000 mg/L for butenafine, from 0.031 to 16.000 mg/L for amphotericin B and natamycin, and from 0.5 to 256.0 mg/L for fluconazole. Following incubation at 35 degrees C for 48 h, minimal inhibitory concentration (MIC) was determined according to the CLSI M38-A document. For amphotericin B and natamycin, the MIC was defined as the lowest drug concentration that prevented any discernible growth. For butenafine and fluconazole, the MIC was defined as the lowest concentration in which an approximately 75% reduction compared to the growth of the control was observed. Candida parapsilosis ATCC22019 was used as quality control strains to validated the results. Mean MIC and MIC range, the MIC at which 50% of the isolates tested were inhibited (MIC(50)) and the MIC at which 90% of the isolates tested were inhibited (MIC(90)), were provided for all the isolates tested by using descriptive statistical analysis with the statistical SPSS package (version 13.0). RESULTS: MIC(90) of butenafine, natamycin, amphotericin B and fluconazole were 4, 8, 2 and 512 mg/L for Fusarium spp., respectively; 0.063, 32.000, 2.000 and 256.000 mg/L for Aspergillus spp., respectively; 0.5, 8.0, 2.0 and 128.0 mg/L for Alternaria alternate, respectively; 0.125, 2.000, 0.500 and 4.000 mg/L for Curvularia lunata, respectively; and 1, 4, 1 and 256 mg/L for unusual ocular pathogens, respectively. CONCLUSIONS: Butenafine exhibits potent antifungal activity against a wide variety of ocular pathogenic fungi, especially for Aspergillus spp., Alternaria alternata, Curvularia lunata, and some unusual ocular pathogens and may have a role in future studies of antifungal eye drops and treating fungal keratitis.

[Comparison of the activities of silver nitrate with those of three antifungal agents against ocular pathogenic fungi in vitro].

OBJECTIVE: To investigate antifungal activity of silver nitrate compared with fluconazole, ketoconazole and amphotericin B against ocular pathogenic fungi in vitro. METHODS: It was an experimental study. Susceptibility tests were performed against 260 isolates (15 genera and 29 species) of ocular pathogenic fungi by broth dilution antifungal susceptibility testing of filamentous fungi (M38-A) approved by National Committee for Clinical Laboratory Standards (NCCLS). Final concentrations ranged from 0.031 to 16.000 mg/L for silver nitrate, ketoconazole and amphotericin B, from 0.5 - 256.0 mg/L for fluconazole. Minimum inhibitory concentration (MIC) was defined as the lowest drug concentration that showed absence of growth or complete growth inhibition (100%). The end points were determined as 100% growth inhibition for silver nitrate and amphotericin B, and > or = 75% growth inhibition for ketoconazole and fluconazole. RESULTS: The MICs at which 90% of isolates were inhibited (MIC(90)) of silver nitrate, ketoconazole, amphotericin B and fluconazole were 2.000, 512.000, 32.000 and 2.000 mg/L for Fusarium species, respectively; 1.000, 256.000, 2.000 and 2.000 mg/L for Aspergillus species, respectively; 2.000, 128.000, 4.000 and 2.000 mg/L for Alternaria alternate, respectively; 2.000, 4.000, 0.125 and 0.500 mg/L for Curvularia lunata, respectively; and 1.000, 256.000, 1.000 and 1.000 mg/L for unusual ocular pathogens, respectively. Silver nitrate was highly active against Aspergillus species (92.9% susceptible at a MIC of < or = 1.0 mg/L) and Fusarium species (96.3% susceptible at a MIC of < or = 2.0 mg/L). 95.6% of Fusarium species and 90.8% of Aspergillus species exhibited resistance to fluconazole, 44.1% of Fusarium species and 42.9% of Aspergillus species exhibited resistance to amphotericin B, 66.2% of Fusarium species exhibited resistance to ketoconazole. The activity of silver nitrate against the fluconazole-resistant, ketoconazole-resistant and amphotericin B-resistant strains was high. CONCLUSION: Silver nitrate has promising activity against a wide variety of ocular pathogenic fungi in vitro, and may have a role in future studies of antifungal eye drops and treating fungal keratitis.

Agrobacterium-mediated transformation of herbicide resistance in creeping bentgrass and colonial bentgrass.

Embryogenic calli were induced from the seeds of creeping bentgrass (Agrostis palustris Huds.) cv. Regent and colonial bentgrass (Agrostis Tenuis Sibth. Fl. Oxen.) cv. Tiger. The embryogenic calli were precultured on fresh medium for 4-7 days and then co-cultivated with Agrobacterium tumefaciens, LBA4404, which contains plasmid vector-pSBGM harboring bar coding region, synthetic green fluorescent protein (sGFP) coding region and matrix attachment region (MAR). After 3 days of co-cultivation, the calli were washed thoroughly and transferred to MS medium containing 2 mg/L of 2, 4-D, 12-15 mg/L phosphinothricin (PPT) and 250 mg/L of cefotaxime. After 2-3 months of selection, the actively growing calli of 'Regent' and 'Tiger' were transferred to MS medium with 12-15 mg/L PPT and 250 mg/L cefotaxime for regeneration. The putative transformants were maintained on MS medium with 3 mg/L PPT for long period but control died within 1 month. After establishing in greenhouse, the transformants also showed strong resistance to 0.4% of herbicide Basta but control plants died within 2 weeks. Under confocal microscope, both young leaves and roots showed significant GFP expression. PCR analysis revealed the presence of a DNA fragment of GFP gene at the expected size (380 bp) in the transformants and its absence in a randomly selected control plant.